Fertilization and syncytia formation in bones, muscles and placenta; exocytosis and enveloped virus infections all involve protein-mediated fusion of two membranes into one. Most of what we know about fusion derives from studying how well characterized viral fusion proteins (e.g., influenza virus hemagglutinin, HA) open fusion pores connecting aqueous compartments. Our recent work has been focused on studying the later stages of an expansion of these pores leading in the case of cell fusion to the complete loss of the membranes that separate two cells and in the case of viruses to delivery of the relatively bulky viral genome into the host cell. Our studies on HA-mediated fusion indicate that low-pH?activated HAs not only initiate fusion by catalyzing early fusion intermediates but also provide the driving force for the entire fusion reaction by bending the contacting membranes out of their initial shape. This latter function: driving the expansion of a fusion pore, is shared by many different fusion proteins and represents the most demanding part of their job. Our work is aimed at the exploration of the mechanisms by which activated fusion proteins control progression of diverse fusion reactions towards their completion. The work can be divided into 2 projects. 1. Involvement of fusion proteins located outside of the contact zone in fusion pore expansion. To formulate the possible mechanism by which proteins expand pores we compared fusion with another kind of membrane remodeling, namely fission of one membrane into two. Based on the literature it has appeared that proteins driving membrane merger in fusion and fission do it in radically different ways. Fission can and likely is mediated by proteins, which are not located between merging membranes. In contrast, fusion has been generally believed to result from the local action of only those fusion proteins, which are located in the contact zone between the membranes and interact directly with the target membrane. However the role of the fusion proteins outside of the contact zone has never been tested. In our work we assessed the role of these ?outsider? proteins in influenza virus hemagglutinin mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. HA-cells and bound RBCs establish extended CZ with area on the order of tens of square microns that are characterized by a relatively constant intermembrane distance of ~13 nm that is close to the height of the HA ectodomain. Readily distinguishable pools of insider and outsider HAs in this system facilitate characterization of their relative fusogenic activity. To selectively inhibit or enhance the actions of hemagglutinin outsiders, antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres (20 nm-, 100 nm- and 2 mm- diameters) too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The mechanisms by which fusion proteins that at the time of the activation are located outside of the contact zone influence fusion remain to be understood. Functional role of HA outsiders in our experiments might indicate that as in the case of vacuolar fusion most of the HA-mediated fusion events develop along the circumference rather than in the central region of the CZ. If so, HA outsiders located near the periphery of the CZ can be important for fusion. Alternatively, HA outsiders do not need to be in the immediate proximity to the fusion site and drive opening and expansion of a fusion pore by generating tension in membrane bilayer. The discovered functional role of fusion proteins located outside of the contact zone goes against the accepted view that fusion is a highly localized phenomenon driven only by a few fusion proteins in the contact zone that directly interact with the target membrane. At the same time, our results rationalize the interesting reports that the entry of many viruses and exocytotic fusion are inhibited by macromolecules, which are added to pre-docked membranes and are, apparently, too large to rapidly enter the tight contact zone. Our finding also strengthens a tempting hypothesis that the oppositely directed processes of membrane fusion and fission work according to a common principle: the proteins drive membrane remodeling from outside of the zone of the actual membrane rearrangement. 2. Mechanistic dissection of tissue-specific eff-1-mediated cell fusion in C. elegans. To study the mechanisms of cell fusion during development of multi-cellular organisms we have focused on the relatively well-characterized fusion in C. elegans. Normal development of C. elegans involves numerous cell fusions with nearly one third of all the nuclei in the adult located in the syncytia. Genetic screens for mutations that inhibit cell fusion within epithelia of C. elegans led to identification of the gene eff-1 (epithelial fusion failure) that encodes type-I membrane proteins EFF-1 expressed as cells become fusion-competen. EFF-1 earlier found to be required for cell fusion is now demonstrated to be sufficient. To dissect the pathway of cell fusion during embryonic development of C. elegans, we developed a new system to simultaneously record, measure and analyze individually fusing epidermal cells in live embryos. In contrast to studies on simpler fusion systems that investigate maximum pore sizes of a few nanometers (microfusion); here we measure the kinetics of large expanding gaps, of the order of hundreds of nanometers/microns (macrofusion), resulting from single cell-cell fusions critical for animal development. We have found that at these scales, each fusion event follows sigmoidal kinetics in wild-type and idf-1 mutant embryos that have Irregular Dorsal Fusion. From the sigmoids, we define lag and macrofusion times as the kinetic parameters for each pair of fusing cells. We found that 9?C incubations block microfusion but not embryonic elongation and idf-1 mutations either block early cell fusion steps or slow down macrofusion rates (Gattegno et al., Submitted). Dissection of cell fusion in a living animal allows us to address the intriguing questions of when, where and how fast cells fuse within tissues. We show that each cell pair within the skin of the C. elegans embryo decides to fuse, or not to fuse, with characteristic kinetic parameters. The functional dissection of eff-1 activity reveals that it acts both in the initiation and expansion of membrane fusion. The emerging research strategy of combining genetic, ultrastructural and kinetic analyses will hopefully be applicable to studying different examples of developmental fusion.